The overall objective of this proposal is to investigate the nature and mechanism of action of the thrombopoietic factors required to bring about the formation of single megakaryocytes and of megakaryocyte colonies from morphologically unrecognizable progenitors, utilizing a mouse marrow cell-plasma clot culture system. Certain factors cause replication and maturation of megakaryocytic precursors (e.g., factors contained in human urinary erythropoietin or in lectin stimulated spleen cell conditioned medium); different factors (e.g., platelet derived activity, human urinary regulatory protein) seem to primarily increase the number of cells capable of responding to other thrombopoietic factors, but are not devoid of maturation inducing activity by themselves, while another potentiating factor from adherent bone marrow cell conditioned medium is ineffective by itself. The influence of varying the amounts and types of these factors added to the system will be systematically studied. Factors, with particular emphasis on those found in urinary protein concentrates, platelets and sera, will be isolated and purified. Preincubation of marrow cells with these factors will be employed in order to modulate the number of responding cells in subsequent clot culture. Mice will be pretreated to change their rate of megakaryocytopoiesis (antiplatelet serum, x-rays, bleeding, drugs, platelet transfusion), and the in vitro responses of their marrow cells to standardized factor stimuli will be compared. Utilizing the information gained in this project, the mouse cell-plasma clot system will be further developed into a clinically useful assay of thrombopoietic factors present in human body fluids. Efforts will be made to determine the physiological relevance of these factors. It will be attempted to establish a human megakaryocyte culture system in analogy to the existing mouse model.